Enhanced circulation effector composition and method

ABSTRACT

A liposome composition comprising small, surface-bound effector molecules is disclosed. The liposomes have a surface layer of hydrophilic polymer chains, for enhanced circulation time in the bloodstream. The effector molecules are attached to the distal ends of the polymer chains. In one embodiment, the effector is polymyxin B, for treatment of septic shock.

This application is a continuation of U.S. application Ser. No.10/832,636 filed Apr. 26, 2004, now allowed; which is a continuation ofU.S. application Ser. No. 10/438,502 filed May 14, 2003, now pending;which is a continuation of U.S. application Ser. No. 09/877,978 filedJun. 8, 2001, now U.S. Pat. No. 6,586,002; which is a continuation ofU.S. application Ser. No. 08/480,332 filed Jun. 7, 1995, now U.S. Pat.No. 6,180,134; which is a continuation-in-part of U.S. application Ser.No. 08/316,436 filed Sep. 29, 1994, now abandoned; which is acontinuation-in-part of U.S. application Ser. No. 08/035,443 filed Mar.23, 1993, now U.S. Pat. No. 6,326,353; all of which are incorporatedherein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to an enhanced-circulation effectorcomposition and method for treating a subject with small effectormolecules which are normally subject to rapid renal clearance from thebloodstream.

BACKGROUND OF THE INVENTION

A number of emerging or current therapies involve intravenous injectionof small (less than 50 Kdaltons) protein, polypeptide or polysaccharideeffectors. Such effectors can include F_(ab) antibody fragments for usein active immunity, cytokines and cellular growth factors forstimulating immunological inflammatory responses, hormones, andpolysaccharides, which are capable of interacting with endothelial cellreceptors to competitively block neutrophil binding to activatedendothelial cells lining the blood vessel (Katre, N. V., et al., Proc.Natl. Acad. Sci. USA 84:1487-1491 (1987); Philips, M. L., et al.,Science 250:1130-1132 (1990); Waldmann, T. A., Annu. Rev. Immunol.10:675-704 (1992)).

Other small polypeptide effectors have been proposed for use in blockingviral infection of target cells in the blood, such as a CD4+glycopeptide which is effective to inhibit binding of humanimmunodeficiency virus (HIV) toCD4^(+ cells (Capon, D. J. and Ward, R. H. R.,) Ann. Rev. Immunol.9:649-678 (1991); Janeway, C. A., Ann. Rev. Immunol. 10:645-674 (1992)).

Polymyxin B, a small basic peptide which is rapidly excreted by thekidneys, is known to react with and neutralize gram-negative bacterialendotoxins, specifically E. coli 0111:B4 liposaccharide (LPS) (Baldwin,G., et al., J. Infect. Diseas. 164:542-549 (1991)). It is not oftenadministered parenterally as a treatment for septic shock syndrome,because high doses of polymyxin B are required for effective treatment.High doses can be fatal, due to renal toxicity, making advanced stagesof septic shock difficult to treat.

The problem of rapid renal clearance observed with polymyxin B is alsoapplicable to other small peptides, such as those discussed above, whichhave been used for parenteral treatment of disease. In general,circulating proteins which are smaller than about 50-60 Kdaltons will becleared by the kidneys with a lifetime of less than 1-2 hours.

In some cases, peptide molecular weight can be increased above thethreshold 50-60 Kdalton size by derivatizing the peptide withbiologically compatible polymers, such as polyethyleneglycol (PEG)(e.g., U.S. Pat. No. 4,179,337). However, this strategy may not alwaysbe effective for small effectors, e.g., those with molecular weightsless than about 5-10 Kdalton. Moreover, derivatizing a polypeptide witha plurality of PEG chains may destroy or reduce the polypeptideactivity, and/or mask key activity sites of the polypeptide.

SUMMARY OF THE INVENTION

The invention includes, in one aspect, a liposome composition for use intreating a subject with a polypeptide or polysaccharide effector whichis effective as a pharmacological agent when circulating in free form inthe bloodstream, but which is rapidly removed from the bloodstream byrenal clearance in free form. The composition includes liposomes havingan outer surface layer of polyethylene glycol chains and the effectorcovalently attached to the distal ends of the chains. A preferredpolymer is polyethylene glycol having a molecular weight between about1,000 and 10,000 daltons.

Preferred effectors include:

(a) an antibody F_(ab) fragment having neutralizing activity against agiven pathogen present in the bloodstream, for use in treating thesubject for infection by the pathogen;

(b) a CD4 glycoprotein, for use in treating the subject for infection byhuman immunodeficiency virus (HIV);

(c) a cytokine or a cellular growth factor, for use in stimulating animmune response in the subject;

(d) a polysaccharide which binds to endothelial leukocyte adhesionmolecule (ELAM), for use in treating inflammation related to neutrophilrecruitment and tissue infiltration;

(e) IL-1 inhibitor or IL-1RA, for treating a subject to achieveimmune-response suppression;

(f) polymyxin B or polymyxin B decapeptide, for treating the subject forseptic shock;

(g) a peptide hormone, for treating a subject to regulate cellulargrowth; and

(h) a peptide, for inhibiting a ligand-receptor cell-binding event.

In one specific embodiment, the invention includes a method ofpreventing progression of gram-negative bacteremia to septic shock and amethod of treating acute septic shock by administering to a subject, aliposome composition containing liposomes having an outer layer ofpolyethylene glycol (PEG) chains and polymyxin B attached to the distalends of the polymer chains.

In another aspect, the invention includes a liposome composition for usein preventing rapid removal from the bloodstream of a polypeptide orpolysaccharide effector by renal clearance. The composition includesliposomes having an outer layer of polyethylene glycol chains, andattached to the distal ends of the chains, is one of the above effectors(a)-(h). These and other objects and features of the invention willbecome more fully apparent when the following detailed description ofthe invention is read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show steps for the synthesis of a maleimide of a DSPEcarbamide of polyethylene glycol (PEG) bis(amine);

FIG. 2 shows steps for the synthesis of a disulfide linkage-containingpropionamide of a DSPE (distearyl phosphatidylethanolamine) carbamide ofpolyethylene glycol (PEG) bis(amine);

FIG. 3 shows a synthetic scheme for the preparation of a PEG-derivatizedPE (phosphatidylethanolamine) containing a terminal aldehyde group;

FIG. 4 illustrates a synthetic scheme for forming a PEG-derivatized DSPEhaving a reactive maleimide group at the PEG terminus;

FIG. 5 illustrates an exemplary method for forming a PEG-derivatizedDSPE containing a bromoacetamide group at the polymer end;

FIG. 6 shows an exemplary method for synthesizing a derivatized DSPElipid with a PEG chain functionalized to contain a terminal hydrazidegroup;

FIGS. 7A-7D show steps in the synthesis of a PEG-derivatized DSPE lipidcontaining a reactive group at the polymer end (FIG. 7A) which can beused to couple to a variety of amine containing groups (7B-7D);

FIG. 8 shows one synthetic approach for forming DSPE derivatized by aPEG spacer chain having a terminal hydrazide group (shown in protectedform);

FIG. 9 shows the covalent coupling of a sulfhydryl-containing peptide tothe terminal maleimide group of a DSPE carbamide PEG derivative;

FIG. 10 shows the covalent coupling of a sulfhydryl-containing peptidevia formation of a disulfide bond to a DSPE carbamide of a terminallyfunctionalized PEG containing a reactive disulfide linkage derived fromSPDP (N-succinimidyl 3-(2-pyridyldithio)-propionate);

FIG. 11 shows the covalent coupling of a peptide through the aldehydegroup of an ethylene-linked derivative of DSPE carbamide of PEG byreductive amination;

FIG. 12 shows a plot of a time course of gallium-67 labelled liposomescomposed of hydrazide PEG-DSPE, partially hydrogenated eggphosphatidylcholine (PHEPC), and cholesterol (PEG-HZ fluid liposomes) orhydrazide PEG-DSPE, hydrogenated serum phosphatidylcholine (HSPC), andcholesterol (PEG-HZ rigid liposomes) in the bloodstream; and

FIG. 13 shows the amino acid sequences for peptides identified by SEQ IDNOS:1-10, in conventional single-letter amino acid code.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Unless otherwise indicated, the terms below have the following meaning:

“Vesicle-forming lipid” refers to any lipid capable of forming part of astable micelle or liposome composition and typically including one ortwo hydrophobic acyl hydrocarbon chains or a steroid group and maycontain a chemically reactive group, such as an amine, acid, ester,aldehyde or alcohol, at its polar head group.

“Effector” refers to polypeptides, mono or polysaccharides, andglycopeptides. Polypeptides, polysaccharides or glycopeptides may havesizes up to about 50-60 Kdaltons.

II. Effector Composition

The invention includes, in one aspect, a liposome composition for use intreating a subject with a small polypeptide or polysaccharide effectormolecule which is effective as a pharmacological agent when circulatingin free form in the bloodstream, but which is rapidly removed from thebloodstream by renal clearance. The composition includes a liposomalcarrier composed of liposomes having an outer surface layer formed ofhydrophilic polymer chains, e.g., PEG. The effector is attached to thedistal ends of the polymer chains in at least a portion of thederivatized vesicle-forming lipid. The effector is attached to thedistal end of a polymer chain to preserve the biological activity of theeffector, such as behaving as a member of a ligand-receptor bindingpair. The preparation of the composition follows the general proceduresbelow.

A. Lipid Components

The liposomal carrier of the composition is composed of three generaltypes of vesicle-forming lipid components. The first includesvesicle-forming lipids which will form the bulk of the vesicle structurein the liposome.

Generally, these vesicle-forming lipids include any amphipathic lipidshaving hydrophobic and polar head group moieties. Such a vesicle-forminglipid for use in the present invention is one which (a) can formspontaneously into bilayer vesicles in water, as exemplified by thephospholipids, or (b) is stably incorporated into lipid bilayers, withits hydrophobic moiety in contact with the interior, hydrophobic regionof the bilayer membrane, and its polar head group moiety oriented towardthe exterior, polar surface of the membrane.

The vesicle-forming lipids of this type are preferably ones having twohydrocarbon chains, typically acyl chains, and a polar head group.Included in this class are the phospholipids, such asphosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidicacid (PA), phosphatidylinositol (PI), and sphingomyelin (SM), where thetwo hydrocarbon chains are typically between about 14-22 carbon atoms inlength, and have varying degrees of unsaturation. The above-describedlipids and phospholipids whose acyl chains have varying degrees ofsaturation can be obtained commercially or prepared according topublished methods. Other suitable lipids include glycolipids and sterolssuch as cholesterol.

The second general component includes a vesicle-forming lipid which isderivatized with a polymer chain. Vesicle-forming lipids for use as thesecond general vesicle-forming lipid component (e.g., are suitable forderivatization with a polymer) are any of those described for the firstgeneral vesicle-forming lipid component. Vesicle forming lipids withdiacyl chains, such as phospholipids, are preferred. One exemplaryphospholipid is phosphatidylethanolamine (PE), which provides a reactiveamino group which is convenient for coupling to the activated polymers.An exemplary PE is distearyl PE (DSPE).

A preferred polymer for use in forming the derivatized lipid componentis polyethyleneglycol (PEG), preferably a PEG chain having a molecularweight between 1,000-10,000 daltons, more preferably between 2,000 and5,000 daltons. Other hydrophilic polymers which may be suitable includepolyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline,polyhydroxypropyl methacrylamide, polymethacrylamide andpolydimethylacrylamide, polylactic acid, polyglycolic acid, andderivatized celluloses, such as hydroxymethylcellulose orhydroxyethylcellulose.

Additionally, block copolymers or random copolymers of these polymers,particularly including PEG segments, may be suitable. Methods forpreparing lipids derivatized with hydrophilic polymers, such as PEG, arewell known e.g., as described in co-owned U.S. Pat. No. 5,013,556.

The third general vesicle-forming lipid component is a lipid anchor bywhich the effector is anchored to the liposomes, through a polymer chainin the anchor. Additionally, the effector is positioned at the distalend of the polymer chain in such a way so that the biological activityof the effector is not lost. The lipid anchor has a hydrophobic moietywhich serves to anchor the lipid in the outer layer of the liposomebilayer surface, a polar head group to which the interior end of thepolymer is covalently attached, and a free (exterior) polymer end whichis or can be activated for covalent coupling to the effector. Methodsfor preparing lipid anchor molecules of this types are described below.

B. Liposome Preparation

The liposomes may be prepared by a variety of techniques, such as thosedetailed in Szoka, F., Jr., et al., Ann. Rev. Biophys. Bioeng. 9:467(1980). Multilamellar vesicles (MLVs) can be formed by simple lipid-filmhydration techniques. In this procedure, a mixture of liposome-forminglipids of the type detailed above dissolved in a suitable organicsolvent is evaporated in a vessel to form a thin film, which is thencovered by an aqueous medium. The lipid film hydrates to form MLVs,typically with sizes between about 0.1 to 10 microns.

The lipid components used in forming the liposomes are preferablypresent in a molar ratio of about 70-90 percent vesicle forming lipids,1-25 percent polymer derivatized lipid, and 0.1-5 percent lipid anchor.One exemplary formulation includes 50-70 mole percent underivatized PE,20-40 mole percent cholesterol, 0.1-1 mole percent of a PE-PEG (3500)polymer with a chemically reactive group at its free end for effectorcoupling, 5-10 mole percent PE derivatized with PEG 3500 polymer chains,and 1 mole percent α-tocopherol.

The liposomes are preferably prepared to have substantially homogeneoussizes in a selected size range, typically between about 0.03 to 0.5microns. One effective sizing method for REVs and MLVs involvesextruding an aqueous suspension of the liposomes through a series ofpolycarbonate membranes having a selected uniform pore size in the rangeof 0.03 to 0.2 micron, typically 0.05, 0.08, 0.1, or 0.2 microns. Thepore size of the membrane corresponds roughly to the largest sizes ofliposomes produced by extrusion through that membrane, particularlywhere the preparation is extruded two or more times through the samemembrane. Homogenization methods are also useful for down-sizingliposomes to sizes of 100 nm or less (Martin, F. J., in SPECIALIZED DRUGDELIVERY SYSTEMS-MANUFACTURING AND PRODUCTION TECHNOLOGY, (P. Tyle, Ed.)Marcel Dekker, New York, pp. 267-316 (1990)).

C. Effector Component

The effector in the composition is a therapeutic polypeptide, mono orpolysaccharide, or glycopeptide characterized, when administeredintravenously in free form, by rapid clearance from the bloodstream,typically within 1-2 hours. The effector itself is effective as apharmacological agent when circulating in free form in the bloodstream.Below are described preferred effectors for use in the invention.

1. F_(ab) Fragment. The F_(ab) fragment is one which has neutralizingactivity against a given pathogen. The composition is used as a passivevaccine effective to provide humoral immunity against one of a varietyof selected pathogenic antigens.

F_(ab) fragments of neutralizing antibodies can be prepared according toconventional methods (Harlow, E., et al., in ANTIBODIES: A LABORATORYMANUAL, Cold Spring Harbor Press, Plainville, N.Y., (1988)). Thefragment is preferably from a humanized monoclonal antibody (M_(ab)).Such antibodies can be prepared by published recombinant DNA methods(Larrick, J. W., et al., Methods in Immunology 2:106 (1991)). Theantibody is preferably coupled to liposomal hydrophilic polymer groupsvia sulfhydryl linkages, as described above.

2. CD4 Glycoprotein Effector. The CD4 glycopeptide is a region of theCD4 receptor of CD4+ T cells (Capon and Ward). The effector acts toblock HIV infection of CD4+ T cells by blocking gp120-mediated HIVbinding to the CD4 receptor. The effector can be produced according toknown recombinant methods (Maniatis, T., et al., in MOLECULAR CLONING: ALABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989)).

3. Cytokines. The cytokines given in Table 1 below are examples ofcytokines which are useful in the present invention. The cytokines maybe obtained by recombinant production methods, according to publishedprocedures. The therapeutic uses of the individual cytokines have beendescribed in the literature (see, for example, Abbas, A. K., et al., inCELLULAR AND MOLECULAR IMMUNOLOGY, W. B. Saunders Company Harcourt BraceJovanovich, Philadelphia (1991)). Some cytokine effectors may beadministered on a short term basis to enhance a weak immunogenic or weakmicrobicidal response. The effectors may be administered on a long termbasis as part of a therapy treatment for cancer or AIDS (Waldmann).TABLE 1 CYTOKINE POLYPEPTIDE SIZE A. Mediators of Natural ImmunityIFN-alpha 18 kD (monomer) IFN-beta 20 kD (monomer) Tumor necrosis factor(TNF) 17 kD (homotrimer) Interleukin-1 (alpha and beta) 17 kD (monomer)Interleukin-6 26 kD (monomer) Interleukins-8's 8-10 (monomer or dimer)B. Mediators of Lymphocyte Activation, Growth and DifferentiationInterleukin-2 14-17 kD (monomer) Interleukin-4 20 kD (monomer)Transforming growth factor (beta) 14 kD (monomer or dimer) C. Mediatorsof Effector Cell Adhesion Gammma Interferon 21-24 kD (homodimer)Lymphotoxin 24 kD (homotrimer) Interleukin-5 20 kD (monomer) D.Mediators of Immature Leukocyte Growth and Differentiation Interleukin-320-26 kD (monomer) Granulocyte-macrophage Colony 22 kD (monomer)Stimulating Factor Macrophage Colony Stimulating 40 kD (dimer) FactorGranulocyte CSF 19 kD (monomer) Interleukin-7 25 kD (monomer)

4. ELAM-1 Binding Inhibitor. Inflammation causes the expression of apolypeptide, endothelial leukocyte adhesion molecule-1 (ELAM-1), on thesurface of endothelial cells of blood vessels, adjacent to sites ofinflammation. ELAM-1, in turn, recognizes and binds a polysaccharidemoiety, sialyl Lewis^(x), on surfaces of neutrophils and recruitsneutrophils to sites of inflammation. By preventing the recognition andbinding of neutrophils by ELAM-1, excessive inflammatory responses dueto conditions such as reperfusion injury, septic shock, and chronicinflammatory diseases, can be avoided.

In this embodiment, the effector is the tetrasaccharide, sialylLewis^(x), recognized by ELAM-1 (Phillips, M. L., et al., Science250:1130-1132 (1990)), for therapeutical use in preventing excessiverecruitment of neutrophils to sites of inflammation in the blood stream.The effector is produced by the glycosylation mutants of Chinese hamsterovary (CHO) cells, and may be obtained in purified form from thecultured cells (Phillips). Alternatively, the effector is produced bychemical and/or enzymatic synthesis (Borman, S., Chem. Eng. News,December 7:25-28 (1992); Ichikawa, Y. et al., J. Am. Chem. Soc.114:9283-9298 (1992)).

5. Inhibitors of IL-1 Activity. The effector in this embodiment is anIL-1 inhibitor, or IL-1 receptor antagonist (IL1PA), which blocksbinding of IL-1 to receptors on lymphocyte cell surfaces (Stylianou, E.,et al., J. Biol. Chem. 267:15836-15841 (1992)).

IL-1 production is stimulated by both endotoxins which cause septicshock and exotoxins which cause toxic shock syndrome (Dinarello, C. A.,Blood 77(8):1627-1650 (1991)). IL-1 production during septic shock ortoxic shock may exacerbate the clinical symptoms observed in patients.Therefore, use of an IL-1 inhibitor effector to decrease the clinicalsymptoms associated with either toxic shock or septic shock may bebeneficial.

IL-1 inhibitor is a 52 to 66 Kd polypeptide that binds specifically toIL-1 to inhibit its immunostimulatory responses. IL1RA is a 23 to 25 KDpolypeptide that competes with binding of IL-1 to its cell surfacereceptors to inhibit IL-1's immunostimulatory responses.

6. Polymyxin B. This effector is a cationic detergent with a hydrophobicportion (6-methyloctanoyl) and a short basic decapeptide portion.Polymyxin B reacts with and neutralizes gram-negative bacterialendotoxins, specifically E. coli 0111:B4 liposaccharide (LPS) (Baldwin,et al.). Polymyxin B is used in the treatment of gram-negative bacterialinfections. Since polymyxin B must be administered frequently and inhigh doses because of its rapid clearance from the bloodstream, itcauses severe irreversible kidney damage. Polymyxin B can be chemicallysynthesized or isolated from spore-forming gram-positive bacilli, suchas Bacillus polymyxa.

Alternatively, the effector is an 11.8 Kdalton peptide isolated fromamebocytes of Limulus polyphemus, limulus antilipopolysaccharide factor(LALF). LALF neutralizes meningococcal lipooligosaccharide, as well asother gram-negative endotoxins, and can be used to treat gram-negativesepsis (Wainright, N. R., et al., In CELLULAR AND MOLECULAR ASPECTS OFENDOTOXIN REACTIONS (Nowotny, A., et al., Eds.) Elsevier SciencePublishers B. V., p. 315 (1990)).

7. Peptide Hormone. This effector can be used in the treatment ofvarious diseases. In one embodiment, the effector is parathyroid hormone(PTH) which is 84 amino acids in length and can inhibit osteoblastdivision. Certain bone cancers are characterized by uncontrolledosteoblast division (Kano, J., et al., Biochem. Biophys. Res. Comm.179:97-101 (1991)). Alternatively, the peptide hormone can be used totarget a liposome to cells that contain receptors for a specific peptidehormone.

D. Attachment of Effector to Liposome Carrier

For effector attachment to liposome carriers, the free polymer end of alipid anchor is activated prior to effector coupling. In the followingspecific examples, both lipid anchor formation and activation reactionsare described. The reactions are shown with respect to the free lipid,either distearylphosphatidyl-ethanolamine (DSPE) or PE.

The activated lipid anchors are then incorporated into liposomalcarriers, as described above.

One advantage of activating the PEG terminal group of the lipid anchorprior to liposome formation is that a broader range of reaction solventsand reaction conditions may be employed. Further, the liposomesthemselves are not exposed to the activating reagents. Thus, the need toremove reagent contaminants from the liposomes is avoided.

It will also be appreciated that the activation reactions may beperformed after lipid anchor incorporation into liposomal carriers. Insome coupling reactions it may be more desirable to activate theterminal PEG groups on preformed liposomes. One advantage of thisapproach is that the activation reaction is confined to the outer,surface-accessible lipids, and thus the activated groups can becompletely quenched prior to use of the composition in therapy. Theapproach is also preferred for reactions in which the activated PEGtermini are unstable in water.

FIGS. 1A-1B show the synthesis of DSPE derivatized with a PEG chain andhaving an activated maleimide group at the chain's free end. Initially,PEG bis(amine) (compound I) is reacted with 2-nitrobenzene sulfonylchloride to generate the monoprotected product (compound II). CompoundII is reacted with carbonyl diimidazole in triethylamine (TEA) to formthe imidazole carbamide (e.g., urea) of the mono2-nitrobenzenesulfonamide (compound III).

Compound III is reacted with DSPE in TEA to form the derivatized PElipid protected at one end with 2-nitrobenzyl sulfonyl chloride. Theprotecting group is removed by treatment with acid to give the DSPE-PEGproduct (compound IV) having a terminal amine on the PEG chain. Reactionwith maleic anhydride gives the corresponding end-functionalized product(compound V), which on reaction with acetic anhydride gives the desiredDSPE-PEG-maleimide product (compound VI). Details of the reactions aregiven in Example 1.

The compound is reactive with sulfhydryl groups, for couplingpolypeptides through a thioether linkage, as illustrated in FIG. 9.

FIG. 2 illustrates an exemplary synthesis of another derivatized lipiduseful for coupling to sulfhydryl-containing polypeptides. Here theDSPE-PEG lipid (compound IV) described above is treated withN-succinimidyl-3-(2-pyridyldithio)propionamide, SPDP, (compound VII) toform the anchor DSPE-PEG lipid (compound VIII). The compound can, forexample, react with a sulfhydryl group of a peptide to thereby couplethe peptide to the lipid through a disulfide linkage as illustrated inFIG. 10.

Another synthetic approach for coupling a protected polyalkylether to alipid amine is shown in FIG. 3. In this reaction scheme, PEG (compoundIX) is initially protected at one of its terminal OH ends by atrimethylsilyl group, as shown at the top of FIG. 3. The monoprotectedPEG (compound X) is reacted with the anhydride of trifluoromethylsulfonate to activate the free PEG end with trifluoromethyl sulfonate(compound XI). Reaction of the activated PEG compound with a lipidamine, such as PE, in the presence of triethylamine, and release of thetrimethylsilyl protecting group by acid treatment, gives the PE-PEGderivative (compound XII). This compound contains a terminal alcoholgroup which is then oxidized in the presence of dimethylsulfoxide (DMSO)and acetic anhydride to form an aldehyde group (compound XIII) which canbe coupled to a peptide via reductive amination, as illustrated in FIG.11. Reaction details are given in Example 2.

More generally, the derivatized lipid components can be prepared toinclude a lipid-polymer linkage, such as a peptide, ester, or disulfidelinkage, which can be cleaved under selective physiological conditions,such as in the presence of peptidase or esterase enzymes or reducingagents, such as glutathione, present intracellularly.

An alternative general method for preparing lipid derivatives of PEGsuitable for coupling to effector molecules involves usingα-amino-ω-carboxy derivatives of PEG (such as compound XIV) as startingmaterials. This alternative approach is illustrated in FIGS. 4, 5, and6.

Methods for preparing heterobifunctional PEG derivatives such ascompound XIV have been described by Zalipsky, S., et al., PolymerPreprints 27(1):1 (1986); Zalipsky, S., et al., J. Bioactive Compat.Polym. 5:227 (1990)). In the reaction scheme shown in FIG. 4, anα-amino-ω-carboxy functionalized PEG (Zalipsky, et al., 1986) is reactedwith N-(γ-maleimidobutyryl-oxy)succinimide ester (GMBS, Pierce), usingan excess of GMBS. The terminal carboxyl group of the resultingmaleimido-PEG (compound XV) is then reacted with a lipid amine, such asPE or DSPE, in the presence of N-hydroxysuccinimide, to link the PEG tothe lipid through an amide linkage (compound XVI). The maleimido groupat the “free” end of the polymer is reactive towards thiol-containingligands, proteins, e.g., immunoglobulins and fragments thereof.

A related scheme is illustrated in FIG. 5, which shows introduction of aterminal bromoacetamide group in an α-amino-ω-carboxy-functionalizedPEG. In the approach shown, a derivative of PEG is reacted withbromoacetyl N-hydroxysuccinimide ester. Thebromoacetamido-functionalized PEG is then coupled to a suitable lipidamine, such as PE or DSPE, as above, to form the derivatized lipid(compound XVIII). The bromoacetamide group, being more selective andmore stable than a maleimide group, allows greater flexibility in themethods used for liposome formation and loading.

The reaction scheme shown in FIG. 6 illustrates the preparation of aderivatized lipid in which the free PEG end is functionalized to containa hydrazide. In the reaction illustrated in FIG. 6, anα-hydroxy-ω-carboxylic acid PEG derivative (compound XIX) (Zalipsky, etal., 1990) is esterified with methanol to protect the terminal acidgroup by formation of the corresponding ester (compound XX). Theterminal hydroxyl group is then converted into a functional groupreactive towards primary amines (Zalipsky, S., et al., in POLY (ETHYLENEGLYCOL) CHEMISTRY: BIOTECHNICAL AND BIOMEDICAL APPLICATIONS (J. M.Harris, Ed.) Plenum Press, pg. 347-370 (1992a)), for example, asuccinimidyl carbonate (SC) derivative (compound XXI). This compound isformed, for example, by reacting compound XX with phosgene followed bysubsequent reaction with N-hydroxysuccinimide (Zalipsky, S., et al.,Biotechnol. Appi. Biochem. 15:100 (1992b)). The resulting activated PEGcompound, SC-PEG-C(O)NHCH₂CO₂-Me (compound XXI) reacts with a lipidamine such as PE or DSPE at the reactive succinimidyl carbonate group toform the functionalized lipid, DSPE-PEG-C(O)NHCH₂CO₂-Me (compound XXII).The methyl ester can be readily hydrazinolyzed to yieldDSPE-NHCO₂-PEG-C(O)NHCH₂C(O)—N₂H₃ (compound XXIII), as shown. Thishydrazide-containing PEG-lipid is incorporated into liposomes byconventional methods. The hydrazide group can be used for attachment ofaldehyde or ketone containing effector molecules.

Such carbonyl groups exist or can be easily generated on numerouscarbohydrate containing molecules, e.g. oligosaccharides, nucleotides,low molecular weight glycosides, lectins, immunoglobulins and otherglycoproteins by chemical (periodate oxidation) or enzymatic reactions(galactose oxidase). The linkages formed, hydrazones, are reasonablystable at pH≧7.5, but are cleavable by acid catalyzed hydrolysis atlower pH values. These linkages can be stabilized by reduction, e.g.,with sodium cyanoborohydride. An advantage of this approach is thestability of hydrazide groups which allows the use of a wide array ofliposome formulations and loading protocols.

Alternatively, as illustrated in FIG. 7A, an α-hydroxy-ω-carboxyderivative of PEG (compound XIX) can be coupled to a lipid containing aterminal amino group, e.g., DSPE, by reaction with N-hydroxysuccinimidein the presence of a coupling agent such as dicyclohexylcarbodiimide,DCC. The resulting intermediate, the N-hydroxysuccinimide (NHS) ester ofα-hydroxy-PEG, is then suitable for coupling to an amino-end containinglipid such as DSPE by displacement of the NHS group to form aα-hydroxy-PEG-DSPE conjugate, linked by an amide bond (compound XXIV,FIG. 7A). The α-hydroxy group of PEG can then be further activated, suchas by reaction with disuccinimidyl carbonate (DSC), to form anα-succinimidyl carbonate-PEG-DSPE compound (compound XXV) suitable forcoupling to a variety of compounds containing reactive amino groups.

Preparation of compound XXIV is described in Example 4. Amino-groupcontaining compounds for coupling to such functionalized lipids willalso possess at least one other functional group to which effectormolecules may be attached. The attachment of the effector molecules mayoccur before or after liposome formation.

In one case, as illustrated in FIG. 7B, the SC-PEG-DSPE is reacted with2-aminoethanedithiopyridine. The derivative formed (compound XXVI) canbe used in the following manner. The dithiopyridine group is reactivetowards thiol-containing molecules but is also quite stable under avariety of conditions. Using mild reducing agents, e.g.,β-mercaptoethanol, it is possible to convert the dithiopyridine groupson the liposomes into free thiols, which in turn can be used in variousconjugation procedures involving ligands containing reactive maleimidoor bromoacetate groups or reactive mixed disulfide groups such asdithiopyridine.

In the reaction illustrated in FIG. 7C, the SC-PEG-DSPE is reacted with3-amino-1,2-propanediol, producing a diol terminated PEG-lipid (compoundXXVII). After incorporation into a liposome, the diol can be oxidized(e.g., with periodate) under mild conditions ([I0₄-]≦10 mM, 4° C.) toprovide a reactive aldehyde. The aldehyde containing PEG-liposomes willreact irreversibly with a variety of amino-containing effector moleculesin the presence of a reducing agent such as sodium cyanoborohydride.

In the reaction illustrated in FIG. 7D, SC-PEG-DSPE is coupled to agalactosamine. The galactose residue on the derivatized lipid (compoundXXVIII) can then be enzymatically oxidized by galactose oxidase. Thealdehyde bearing PEG-liposomes obtained by this process can be used forconjugation with amino-group containing effector molecules. In additionto the mildness of the reaction conditions, the aldehyde groups aregenerated solely on the outer surface of the liposome.

Additionally, there is evidence that oxidized galactose residues areuseful for stimulation of the immune system, specifically for T cellactivation. A liposome having oxidized galactose residues on its surfaceis likely to act as an adjuvant and might be useful in vaccines (Zheng,B., et al., Science 256:1560-1563 (1992)).

In another procedure, illustrated in FIG. 8 and described in Example 5,DSPE-PEG-hydrazide is prepared. First PEG is reacted with ethylisocyanatoacetate in the presence of triethylamine to generate mono anddi-end carboxylated species of PEG, where the carboxylic acid functionsare connected to the PEG skeleton via intervening carbamate bonds. Themonocarboxylated species is purified by ion-exchange chromatography onDEAE-Sephadex (compound XXIX, identical to compound XIX). Compound XXIXis reacted with tert-butyl carbazate to generate theω-hydroxy-α-Boc-hydrazide derivative of PEG (compound XXX). The hydroxylterminus of PEG is then activated by reaction with disuccinimidylcarbonate to form compound XXXI prior to reaction with DSPE to generatethe desired lipid-PEG-α-Boc hydrazide product (compound XXXII). CompoundXXXII is deprotected with 4M HCl in dioxane to form the free hydrazidegroup. Lipid-PEG-hydrazide may then be incorporated into liposomes. Thehydrazide groups are reactive towards aldehydes, which as describedabove, can be generated on numerous biologically relevant compounds.

The methods just described may be applied to a variety of lipid amines,including PE, cholesteryl amine, and glycolipids with sugar aminegroups. It will be appreciated that a variety of alternative couplingreactions, in addition to those just described, are suitable forpreparing vesicle-forming lipids derivatized with hydrophilic polymerssuch as PEG, having terminal groups which are activated or are reactivein protein coupling reactions.

1. Maleimide Coupling. Maleimides are widely used protein modifyingreagents and are especially useful when the maleimide is one of twofunctional groups in a heterobifunctional crosslinking reagent. Thereaction of maleimides with sulfhydryl groups involves Michael additionof the mercaptan group to the activated double bond. Reaction with aminogroups occurs by the same mechanism, but at a much slower rate. Sincemercaptan is the most reactive species, particularly at neutral pH, themaleimide group can be used to target a small number of sulfhydrylgroups and good selectivity is usually achieved.

In one preferred embodiment, a derivatized lipid, such as PE- orDSPE-PEG, is prepared to contain a terminal maleimide group (compoundsVI and XVI), as illustrated in FIGS. 1 and 4 above. The lipid, afterincorporation into liposomes, is then reacted with asulfhydryl-containing effector, typically a polypeptide, under suitablecoupling conditions. The reaction of the terminal maleimide-PEG lipid(compound VI or XVI) with a peptide sulfhydryl group is illustrated inFIG. 9. As shown, the reaction couples the protein to the lipid polymerthrough a thioether linkage, to give the derivatized DSPE (compoundXXXIII). Use of this synthetic approach to couple proteins to liposomesis described in Example 6.

The efficiency of β-galactosidase coupling to liposomes containing amaleimide coupling agent in the presence or absence of DSPE-PEG3500 hasbeen examined and the results are discussed below.

Reactions were carried out with liposomes prepared to contain, as themaleimide coupling agent, either (a) the DSPE derivative of succinimidyl4-(p-maleimidophenyl)butyrate (MBP), (b) the DSPE derivative ofN-(11-maleimido-undecanoyl) (C11), or (c) the maleimide of PE-PEG3500.Reactions carried out with (c) are described in detail in Example 6.

After carrying out the protein-liposome coupling reaction, performed asdescribed above for (a)-(c), the amount of liposome-bound enzyme wasquantitated. Recovery of liposomes was measured by scintillationcounting and the recovery of protein was measured by thebeta-galactosidase assay and direct quantitation of the protein amountas described in Example 6.

The maleimide of the DSPE carbamide of PEG3500 was very effective incrosslinking β-galactosidase to liposomes, either in the presence orabsence of DSPE-PEG3500 chains. As seen in Table 2, there wasessentially no difference in the amount of protein crosslinked to eithertype of liposome in two separate experiments. In addition, the amount ofprotein coupled to the PE-PEG maleimide was much higher than to eitherthe MPB or MPB-C₁₁ maleimides.

The presence of “non-activated” DSPE-PEG3500 in the liposomes had littleeffect on the levels of coupling of the protein to DSPE-PEG-maleimideliposomes, but inhibited the level of protein coupling to liposomescontaining either the MPB lipid, or the MBP-C₁₁ lipid. TABLE 2Phenotype″ PEG-DSPE Crosslinker 10 mM 2-ME ng Protein/μmol Lipid* − MPB1609/2284 − MPB + (−80) + MPB (−282)  − C₁₁ 690 − C₁₁ + 847 + C₁₁ 358(−157) + C₁₁ +  80 − 3500 10,033   − 3500 + 572 + 3500 10,765/12,412 +3500 + 110*Background binding in the absence of crosslinker has been subtracted.Background values range from 500-1000 ng protein/μmol lipid. There was atendency for background values to be somewhat (10-30%) higher in thepresence of PEG-DSPE; this may not be significant. Multiple entriesdenote multiple separate crosslinking experiments.

2. Coupling by 3-(2-pyridyldithio)propionamide.

The reaction of dithio propionamides with a sulfhydryl group producescoupling of functionalized lipids to sulfhydryl-containing molecules viaa disulfide linkage. Disulfide exchange occurs readily at pH 8, in anon-reducing environment. The method involves reaction of a thiol groupin a peptide with a liposome prepared to containDSPE-PEG-(2-pyridyldithio) propionamide). The reaction couples theprotein to the liposomes through a disulfide linkage as illustrated inFIG. 10 (compound XXXIV).

3. Reductive Amination

In this approach, the terminal hydroxyl group of a PEG chain, covalentlylinked at one end to PE or DSPE, is converted to the correspondingaldehyde by oxidation under mild conditions. The oxidation step may becarried out before or after incorporation into liposomes to produce thealdehyde form of the derivatized lipid (compound XIII in FIG. 3).Reaction of the aldehyde with the amine group of an effector moleculegives the Schiff base (compound XXXV, FIG. 11) which is then reduced tothe desired derivatized lipid containing an amino-linked peptide(XXXVI).

As indicated above, the polymers can also be activated for effectorcoupling in preformed lipids, i.e., with the polymer-derivatized lipidsalready incorporated into liposomes. One advantage of this approach isthat only polymer moieties on the outer surface of the liposomes areactivated. In one general approach involving PEG polymers, the terminalOH groups are first oxidized by treatment with sodium periodate for 2hours at 20° C. in the dark. After oxidation, the excess reagent isremoved, and the liposomes are incubated with the effector molecule,e.g. F_(ab) fragments, in the presence of 2M sodium cyanoborohydride (10μL/mL) at 20° C. for 14 hours. After completing the incubation, theliposomes can be chromatographed on a Sepharose to remove free(non-linked) effector molecules.

III. Bloodstream and Tissue Retention of Liposomes ContainingEnd-Functionalized PEG-DSPE

In vivo studies were undertaken to determine the bloodstream and tissueretention of liposomes containing end-functionalized PEG-DSPE, asdescribed in Example 7. End-functionalized PEG-DSPE contains achemically active group which can be used for attaching a variety ofcompounds to liposomes. From these studies it has been determined thatend-functionalization does not affect the extended lifetime in thebloodstream of liposomes containing PEG-DSPE, monomethoxy PEG-DSPE, orother similarly modified vesicle-forming lipids.

In experiments performed in support of the present invention, liposomescontaining PEG-DSPE end-functionalized by hydrazide were prepared. Thehydrazide group at the end of a PEG chain can be used for theintroduction of other functional groups, or can be used in numeroustypes of conjugation schemes (Inman, J. K., Meth. Enzymol. 34:30-58(1974)). Particularly useful is hydrazide's reactivity toward variousglycoproteins, such as immunoglobulins (Wilchek, M., and Bayer, E. A.,Meth. Enzymol. 138:429-442 (1987)), for attaching these molecules toliposomes.

Gallium 67-labelled, hydrazide end-functionalized PEG liposomes wereinjected in rats by tail vein injection at about 10-20 micromolarphospholipid/kg body weight. Blood samples were obtained by retroobitalbleeding at defined times. The percent of gallium labelled liposomesremaining in the bloodstream was determined at 0, 15 minutes, 1, 3, 5,and 24 hours and is presented in Table 3. The percent injected gallium67-labelled liposome dose remaining in the blood stream at differenttimes is illustrated in a half log plot versus time in FIG. 12.

After 24 hours the animals were sacrificed and tissues removed for labelquantitation. The percent of the injected dose found in selected tissuesat 24 hours is presented in Table 3.

The blood and tissue retention of Ga-labelled, hydrazideend-functionalized liposomes having two different lipid compositionswere also compared as shown in Table 3. A fluid liposome composition wasprepared from partially hydrogenated egg phosphatidylcholine (PHEPC). Atypical liposome composition contains the hydrazide PEG-DSPE lipid,partially hydrogenated egg PC (PHEPC), and cholesterol in alipid:lipid:lipid mole ratio of about 0.15:1.85:1. A rigid liposomecomposition was prepared by substituting hydrogenated serumphosphatidylcholine (HSPC) for PHEPC at the same mole ratio.

As is indicated in Table 3, the fluidity of the liposome compositiondoes not affect the blood retention time of the liposomes. However, thefluidity of the liposome composition does appear to affect the tissuedistribution of the end-functionalized liposome. For example, rigidliposomes are preferentially retained by liver, spleen and bone tissue.Fluid liposomes are preferentially retained by the kidneys, heart, skinand muscle tissue. TABLE 3 % Injected 67 GA Dose Detected at SpecifiedTimepoints Peg-HZ Rigid PEG-Hz Fluid Blood  0 101.1 ± 12.0  100.2 ± 5.4 15 min. 89.6 ± 11.2  81.6 ± 2.5   1 hr.  84 ± 11.1 81.7 ± 7.4   3 hr. 76 ± 10.5 75.3 ± 5.1   5 hr. 71.7 ± 10.7  66.3 ± 3.8  24 hr. 33.4 ±6.8  34.3 ± 0.68 Tissues at 24 hr. liver 12.1 ± 1.2   8.8 ± 0.81 spleen5.1 ± 0.47  4.7 ± 0.64 kidneys 1.4 ± 0.22  1.7 ± 0.25 heart 0.36 ± 0.0370.77 ± 0.21 lungs .62 ± 0.23 0.58 ± 0.03 skin .086 ± 0.03  0.16 ± 0.08muscle .08 ± 0.03 0.29 ± 0.02 bone .28 ± 0.09 0.04 ± 0.01IV. Therapeutic Effector Compositions

Below are described specific embodiments of the effector composition ofthe invention, and their intended use as injectable therapeutic agents.

A. Compositions for Enhancing an Immune Response

In one general embodiment, the effector in the liposome composition is amolecule capable of enhancing an immune response when administeredparenterally.

1. F_(ab) Effector. The F_(ab) effector composition is used as a passivevaccine to provide humoral immunity against one of a variety of selectedpathogenic antigens. The composition is administered to supplement aweakened immune response to a given antigen.

The vaccine effector composition is administered intravenously shortlyafter exposure to, or shortly before expected exposure to a selectedpathogen. The composition is preferably injected in an amountcorresponding to between about 0.1 to 2 mg antibody/kg body weight.After IV administration, the composition circulates in the bloodstream,at an effective concentration, for 1-2 days.

2. CD4 Glycoprotein Effector. Numerous therapies for the prevention andtreatment of human immunodeficiency virus (HIV) infection and acquiredimmune deficiency syndrome (AIDS) have been proposed. These therapiestarget different steps in the process of viral infection. Frequently,therapy includes the administration of drugs which interfere with viralreplication, such as AZT and DDI. The administration of these drugs isaccompanied by toxic side effects, since the replication process ofnormal cells is also affected.

Another step in the process of viral infection which is targeted intherapy is viral attachment to cells. HIV binds with specificity to theCD4 receptor of CD4+ T cells. By mechanisms not yet fully understood,the CD4+ cells eventually can become infected by HIV. Soluble CD4receptor polypeptides have been administered intravenously toHIV-infected patients to prevent further HIV infection of a patient'sCD4+ T cell population. Heretofore, this therapy has not been effective,since CD4 receptor fragments are rapidly cleared from circulation in theblood stream, and inhibitory plasma concentrations cannot be maintained(Capon and Ward).

The effector molecule in this embodiment is a soluble CD4 receptorpolypeptide capable of binding to the gp120 glycoprotein of humanimmunodeficiency virus (HIV) to prevent binding of HIV to CD4+ T cells.In a preferred embodiment covalent attachment of CD4 is accomplished bycoupling periodate oxidized CD4 with hydrazide group containingliposomes.

CD4 administered as a long-circulating liposomal composition will remainin the blood stream for a longer period of time. The CD4 effectorcomposition can be administered intravenously during early or latestages of HIV infection, most beneficially in combination with otherdrugs used in AIDS therapeutics, so that HIV particles bound to theliposomes, to the extent these are taken up by infected cells, will alsodeliver a dose of the anti-viral agent to the infected cells. AZT andDDI are examples of anti-HIV drugs which may be encapsulated in theliposome compositions.

The liposome composition should be administered intravenously in a doseequivalent to an effective blood stream CD4 concentration of 1-10micromolar. Doses of 5-40 mg CD4/kg body weight can be administered,typically at intervals of 2-14 days between treatments, with the levelof HIV present in the bloodstream being monitored during treatment bystandard assay methods.

Principal advantages of this composition are the increased circulationtime of the CD4 effector in the blood stream and the polyvalentpresentation of the effector on the surface of the liposomes. Improvedaffinities of polyvalent CD4 presentation has recently been described(Chen, L. L., et al., J. Biol. Chem. 266:18237-18243 (1991)). Asdescribed above, CD4 receptor fragments are cleared rapidly by renalfiltration. Covalent attachment of the CD4 polypeptide to liposomalcarriers prevents renal clearance, and permits circulation of thepolypeptide effector composition for 24-48 hours in the blood stream.

Additionally, the polyvalent CD4-bearing liposomes resemble CD4+ T celllymphocytes in that the CD4 glycoproteins are presented on hydrophobicsurfaces which mimic the surfaces of T cell lymphocytes. Thispresentation is likely to serve as a decoy binding HIV particles and HIVinfected cells expressing gp120 so that healthy CD4+ lymphocytes arespared.

3. Effector for Stimulating Inflammatory Immune Responses.

Some medical conditions are treated indirectly, by stimulation of thebody's natural immune response. Such conditions can includeimmunodeficiency diseases, such as AIDS, chronic infectious, and certaintypes of cancers. One immunostimulant therapy involves intravenousinjection of cytokines, which can acts to stimulate B cell and T cellimmune responses in a variety of ways.

The cytokine effector composition may be administered on a short termbasis to enhance a weak immunogenic or weak microbicidal response.Alternatively, the cytokine effector composition may also beadministered on a long term basis as part of a therapy treatment forcancer or AIDS. The effector composition may be administeredintravenously at doses of about 0.5 to 5.0 mg/kg body weight to enhancean immunogenic response. These doses result in an effective cytokineconcentration of about 0.1-1 micromolar in the blood stream.

B. Compositions for Blocking Binding to Cell Receptors

In another general embodiment, the effector in the liposome compositionis a molecule capable of blocking the binding of an endogenous agent toa cell receptor, to achieve a desired therapeutic effect.

1. ELAM-1 Binding Inhibitor. As one example, inflammation causes theexpression of a polypeptide, endothelial leukocyte adhesion molecule-1(ELAM-1), on the surface of endothelial cells of the blood vessels.ELAM-1, in turn, recognizes and binds a polysaccharide moiety onsurfaces of neutrophils, and recruits neutrophils to sites ofinflammation. By preventing the recognition and binding of neutrophilsby ELAM-1, excessive inflammatory responses due to conditions, such asreperfusion injury, septic shock, and chronic inflammatory diseases, canbe avoided.

In this embodiment, the effector is used to prevent the excessiverecruitment of neutrophils to sites of inflammation in the blood stream.The effector is sialyl Lewis^(x) recognized by ELAM-1 (Phillips). Thispolysaccharide effector is covalently attached to long-circulatingliposomal compositions by the methods described above. In a preferredembodiment attachment of sialyl Lewis^(x) to liposomes is accomplishedvia the reducing end of the glucosamine residue. The reducing end caneasily react with a hydrazide group of a DSPE-PEG preparation. Couplingof the polysaccharide to the liposomal carrier composition prevents thepolysaccharide's clearance by the kidney, and maintains an effectiveconcentration of the polysaccharide effector over a 48 hour period. Theliposomal carrier composition is administered in doses of 10 to 50micrograms/ kg body weight in a timely fashion, intravenously, and closeto the site of inflammation.

2. Inhibitor of IL-1 Activity. As a second example, the effector is IL-1inhibitor, which inhibits IL-1's immunostimulatory activity, or IL-1receptor antagonist (IL1RA), which blocks the binding of IL-1 tolymphocyte cell surfaces. These molecules may be administered to asubject for treatment of septic shock, toxic shock, colonicinflammation, or leukemic cell proliferation. In this aspect of theinvention, the liposomal carrier composition is administered in doses of20 to 50 micrograms/kg body weight on a short term basis for thetreatment sepsis, toxic shock or colonic inflammation. The liposomalcarrier composition may also be administered at 1 to 2 day intervals ona long term basis for the treatment of leukemia.

Other molecules effective to block the binding of specific cytokines tospecific lymphocyte populations may also be employed.

The use of the long-circulating effector composition, for use inblocking the binding of endogenous agents to cell receptor sites,provides two advantages over the use of free effector. First, theeffector is maintained in the bloodstream over an extended period, byvirtue of blocking renal clearance of the effector. Secondly, theeffector molecule, in liposome-bound form, provides greater sterichindrance at the cell surface site of the receptor. Also, thecompetitive binding or blocking effector and endogenous agent on thereceptor site is shifted toward the blocking agent, since the liposomalstructure will be displaced at a slower rate kinetically, due to itslarger size and number of blocking molecules in the region of thereceptor site.

3. Peptide Hormones. In this embodiment the effector composition isuseful in the treatment of various diseases that respond to peptidehormones. In one embodiment, the effector is parathyroid hormone (PTH)which is effective to inhibit uncontrolled osteoblast division.

4. Peptide. In this embodiment, the effector is a short peptide that hascell-binding activity and is effective to compete with a ligand for areceptor site. Inhibition of the ligand-receptor cell-binding eventpotentially results in arresting the infection process.

In general, useful peptides will have cell-binding activity due to aportion of sequence other than the end of the peptide. In this way,after attachment to the polymer chain on the liposome, the peptideremains active. Another general feature of useful peptides is theirsmall size. Peptides of between about 4-20 amino acids are preferred.

One exemplary peptide, YIGSR, identified herein as SEQ ID NO:6 (FIG.13), is useful for blocking metastases of tumors. SEQ ID NO:6 is one ofthe peptide sequences in the B1 chain of laminin responsible for theglycoprotein's adhesive properties and is known to bind to the lamininreceptor. Laminin, the protein in which the YIGSR sequence occurs, is aconstituent of basement membranes. Circulating metastatic cells whichover-express the laminin receptor may find their way to lamininmolecules in the basement membrane where they may become attached andestablish metastatic tumors. By introducing exogenous YIGSR, the lamininreceptors of circulating metastatic cells are blocked, therebyinhibiting tumor establishment.

Similarly, the peptide arginine-glycine-aspartic acid-serine (RGDS) hasexperimentally been shown to inhibit the establishment of metastatictumors by interfering with the binding of tumor cells to fibronectin(Humphries, M. J., et al., Science 233:467-469 (1986)). Like YIGSR, RGDSis a peptide sequence involved with tumor cell adhesion to basementmembranes.

The infection of lymphocytes by HIV also involves a specificpeptide-receptor interaction (Nehete, P. N., et al., J. Virol.67:6841-6846 (1993)). Here, the receptor is the CD4 protein and thepeptide is the HIV envelope protein gp120. The peptide binding sequencesare located in the V3 loop of gp120. Several peptide sequences ofbetween 8-15 amino acids have been implicated in the bindinginteractions. These sequences include SEQ ID NO:1 through SEQ ID NO:5and are shown in FIG. 13.

Pseudomonas cepacia infections also exhibit specific binding to thecells they infect (Sajjan, U. S., et al., Inf. Immun. 61:3157-3163(1993)). Pseudomonas pilin proteins, which are found on the bacterialcell surface, act as receptors for host proteins called mucins. Suitablepeptides have been disclosed (e.g., Sastry, P. A., et al., J.Bacteriology 164(2):571-577 (1985); Lee, K. K., et al., MolecularMicrobiol. 3(11):1493 (1989)).

C. Antimicrobial Composition

In this embodiment the effector is a compound which is useful in theprevention and treatment of septic shock. The causal agents of septicshock are endotoxins which accumulate during systemic gram-negativebacterial infections (Jawetz, E., in BASIC AND CLINICAL PHARMACOLOGY(Katzung, B. G., Ed.) Apple & Lange, Los Altos, Calif., pg. 511 (1987)).Because of the rapid onset of severe sepsis, treatment is often notbegun until critical stages of sepsis.

The antimicrobial agent which has been used most successfully intreating septic shock against in cases of septic shock is polymyxin B.Because the compound is rapidly excreted, high doses of polymyxin B arerequired for effective treatment. The high doses, unfortunately, canlead to severe renal toxicity.

In the present invention, polymyxin B circulation in the bloodstream isextended severalfold by its attachment to long-circulating liposomes.The compound is attached to long-circulating liposomal compositioncarriers by the coupling methods described above.

The liposomal composition is administered on a short term basis, at adose of 0.1-0.5 mg/ kg body weight, as a prophylactic for individuals atrisk of, or suffering from acute septic shock. Features of the polymyxinB liposomal composition, already discussed, will minimize polymyxin B'srenal accumulation and renal toxicity.

The following examples illustrate methods for preparing derivatizedlipids and protein-coated liposomes in accordance with the invention.

EXAMPLE 1 Preparation of DSPE-PEG-Maleimide

A. Preparation of the Mono 2-nitrobenzene-sulfonamide of PEG bis(amine)(compound II)

A mixture of 1.7 g (0.5 mmole) of commercially available polyethyleneglycol bis(amine) and 104 mg (0.55 mmole) of 2-nitrobenzene sulfonylchloride were added to a round-bottomed flask. The minimum amount ofdioxane to effect solution (about 15 mL) and 280 microliters oftriethylamine (2 mmole) were added. The reaction flask was stoppered andallowed to stand at room temperature for 4 days.

Thin layer chromatography (TLC) on silica coated plates using a solventmixture of the following composition CHCl₃:CH₃OH:H₂O:NH₄OH, 130:70:8:0.5(v/v/v/v), showed fluorescence quenching spots at R_(f)=0.87 to 0.95 andR_(f)=0.68-0.75. The 2-nitro benzene sulfonyl chloride was a morecompact spot at R_(f)=0.85. The UV absorbing material at R_(f)=0.87-0.95was tentatively identified as the bis-2-nitro-benzenesulfenamide. Thematerial at R_(f)=0.68-0.75 was assigned to the desiredmono-2-nitrobenzenesulfonamide of the starting diamine.

The solvent was evaporated under vacuum to obtain 2.135 g of a yellowsyrup. The crude syrup was dissolved in 5 mL chloroform and placed atthe top of a 21 mm×270 mm column of SiO₂ wetted with chloroform. Theproduct was purified by passing through the column, in sequence: Volume% Volume % MeOH containing Amount (mL) Chloroform 1% conc. NH₄OH 100100% 0% 200 90% 10% 100 80% 20% 100 70% 30%

Fifty mL aliquots were collected separately and assayed by TLC asdescribed above. Most of the yellow, ninhydrin positive-reactingmaterial was eluted in the 20% MeOH fraction. The fractions were driedand resulted in recovery of 397 mg of a bright yellow solid. The yieldof the pure sample was about 20%.

B. Preparation of the Imidazole Carbamide of the Mono2-nitrobenzenesulfonamide of PEG bis(amine)(compound III)

550 mg (0.15 mmole) of the 2-nitrobenzenesulfonamide of PEG bis(amine),compound II, were dissolved in anhydrous benzene. To this was added 49mg of carbonyl diimidazole (0.3 mmole) and 28 microliters (0.20 mmole)of triethylamine. The air in the reaction vessel was displaced withnitrogen, the flask sealed and the reaction mixture was heated in an 80°C. oil bath for 4 hours. TLC on silica-coated plates using the samesolvent system as described above showed that all of the startingsulfonamide (Rf=0.72) had been consumed, and had been replaced by aniodine absorbing material at Rf=0.92. The solvent was removed undervacuum. The residue was dissolved in about 2.5 mL chloroform andtransferred to the top of a 21×280 mm column of silica which was wettedwith chloroform. The following solvents were passed through the column,in sequence: Volume % Volume % MeOH containing Amount (mL) Chloroform 1%conc. NH₄OH 100 100% 0% 100 90% 10% 200 80% 20%

50 mL fractions were collected and assayed by TLC. The desired product,compound III, was found predominantly in the 80-20 chloroform-methanolfractions. Upon evaporating the pooled fractions to dryness, 475 mg of alemon-yellow solid was obtained (compound III).

C. Preparation of the DSPE Carbamide of the 2-Nitrobenzene Sulfonamideof PEG Bis(amine)

To the 450 mg (0.125 mmole) of 2-nitrobenzenesulfonamide of theimidazole carbamide of PEG bis(amine) (compound III) dissolved in 4.5 mLbenzene was added 93 mg DSPE (0.125 mmole) and 70 microliters (0.50mmole) of triethylamine. The reaction flask was then flushed withnitrogen, stoppered, and the contents heated in an oil bath at 80° C.for 6 hours with stirring. The reaction mixture was then cooled to roomtemperature and analyzed by TLC. TLC indicated that all of the DSPE hadbeen consumed (e.g., the reaction had gone to completion). The solventwas evaporated under vacuum and the residue was dissolved in 2.5 mLchloroform and placed at the top of a 21×260 mm column of silica wettedwith chloroform. The sample was purified by passing through the columnin sequence: Volume % Volume % MeOH containing Amount (mL) Chloroform 1%conc. NH₄OH 100 100% 0% 200 90% 10% 100 80% 20% 100 70% 30%

The desired product eluted at 20% (1% conc. NH₄OH in MeOH), wasevaporated and afforded 358 mg of a bright yellow solid with an Rf=0.95.Fractions containing imidazole were not used and the final yield of theproduct (0.0837 mmoles) was 65%.

D. Preparation of the DSPE Carbamide of PEG Bis(amine) (Compound IV)

The product from Example 1C above (˜358 mg) was dissolved in 10 mLethanol. To this solution was added 2.4 mL water and 1.2 mL acetic acid.The mixture was allowed to stand at room temperature for 18 hours. TLCanalysis after 18 hours indicated that only partial deprotection hadoccurred. To the reaction mixture was added another 2.3 mL water and 1.2mL acetic acid and the reaction mixture was then allowed to stirovernight. TLC analysis on silica-coated plates using a similar solventsystem as described above revealed florescence quenching materials withR_(f) values of 0.86 and 0.74, respectively. The desired ninhydrinreactive, phosphate-containing material migrated with an Rf value of0.637. This spot showed no fluorescence quenching.

The solvent was removed under vacuum. The remaining residue wasredissolved in 15 mL chloroform and extracted with 15 mL 5% sodiumcarbonate. The mixture was centrifuged to effect separation, and thesodium carbonate phase was reextracted 2× with 15 mL chloroform. Thecombined chloroform extracts were evaporated under reduced pressure toobtain 386 mg of wax. TLC indicated that the wax was largely a ninhydrinpositive, phosphate containing lipid of R_(f)=0.72.

The wax was dissolved in 2.5 mL chloroform and placed on a silica columnwhich had been wetted with chloroform. The following solvents werepassed through the column in sequence: Volume % Volume % MeOH containingAmount (mL) Chloroform 1% conc. NH₄OH 100 100% 0% 200 90% 10% 100 80%20% 100 70% 30% 100 50% 50% 100 0% 100%

The samples were assayed by TLC. The desired product was found infractions containing 70-30 and 50-50 chloroform-methanol as eluent.These samples were combined and evaporated to dryness under vacuum toafford 91 mg (22 micromoles) of a viscous syrup.

E. Preparation of the Maleic Acid Derivative of the DSPE Carbamide ofPEG Bis(amine) (Compound V)

To 18 micromoles of the viscous syrup prepared in Example 1D above anddissolved in 1.8 mL chloroform was added 3.5 mg (36 micromoles) maleicanhydride and 5 microliters (36 micromoles) triethylamine. The stopperedflask containing the reaction mixture was allowed to stand at roomtemperature for 24 hours and the solvent was subsequently evaporatedunder reduced pressure. TLC on silica plates indicated that all of thestarting material had been replaced by a ninhydrin-negative, phosphatecontaining material of Rf=0.79-1.00 (Compound V).

F. Preparation of the Maleimide of the DSPE Carbamide of PEG Bis(amine)(Compound VI)

The syrup was dissolved in 2 mLs acetic anhydride saturated withanhydrous sodium acetate. The solution was heated in a 50° C. oil bathfor two hours. After cooling to room temperature, 10 mL ethanol wasadded to the contents of the flask and the volatile components were thenevaporated under vacuum. This step was repeated twice to remove excessacetic anhydride and acetic acid. The resulting residue was taken up 1mL chloroform and passed through a silica gel column using the followingsolvents in sequence: Volume % Volume % MeOH containing Amount (mL)Chloroform 1% conc. NH₄OH 100 100% 0% 200 90% 10% 100 80% 20% 100 70%30%

50 mL samples were collected and the main product was found in thefractions eluted with 90-10 chloroform-methanol. The fractions werecombined and evaporated to dryness under vacuum to afford 52 mg of apale yellow viscous oil, which by TLC migrated with an Rf of 0.98 andwas determined to contain phosphate. 12.3 micromoles of product(compound VI) were obtained, corresponding to a yield of about 34%.

EXAMPLE 2 Preparation DSPE-PEG 3-(2-pyridyldithio)propionamide

The DSPE carbamide of PEG bis(amine) (compound IV, 50 micromoles) isdissolved in 3 mL of anhydrous methanol containing 50 micromoles oftriethylamine and 25 mg of N-succinimidyl 3-(2-pyridyldithio)propionate(SPDP, Pierce, Rockford, Ill.). The reaction is carried out at roomtemperature for 5 hours under an argon atmosphere. Methanol is removedunder reduced pressure, and the products are redissolved in chloroformand applied to a 10 mL silica gel column, using silica gel which hasbeen previously activated at 150° C. overnight. A similar solvent systemas described in Example 1 is used to purify the product. Analysis on TLCplates indicates a product (compound VIII) with an R_(f)=0.98 whichreacts negatively with ninhydrin, contains phosphate and has no freesulfhydryl groups. When the product is treated with excessdithiothreitol, 2-thiopyridinone is released.

EXAMPLE 3 Preparation of a PEG-Derivatized PE Containing a TerminalAldehyde Group

A. Preparation of 1-trimethylsilyloxy-PEG (Compound X)

15.0 gm (10 mmoles) of PEG MW 1500, (Aldrich Chemical, St. Louis, Mo.)was dissolved in 80 mL benzene. 1.40 mL (11 mmoles) of chlorotrimethylsilane (Aldrich Chemical Co.) and 1.53 mL (1 mmoles) of triethylaminewas added. The mixture was stirred at room temperature under an inertatmosphere for 5 hours.

The mixture was filtered by suction to separate crystals oftriethylammonium chloride and the crystals were washed with 5 mLbenzene. Filtrate and benzene wash liquids were combined. This solutionwas evaporated to dryness under vacuum to provide 15.83 grams ofcolorless oil which solidified on standing.

TLC of the product on Si-C₁₈ reversed-phase plates using a mixture of 4volumes of ethanol with 1 volume of water as developer, and iodine vaporvisualization, revealed that all the polyglycol 1500 (R_(f)=0.93) hadbeen consumed and was replaced by a material of R_(f)=0.82. An infra-redspectrum revealed absorption peaks characteristic only of polyglycols.

Yield of 1-trimethylsilyloxy-PEG, M.W. 1500 (compound X) was nearlyquantitative.

B. Preparation of Trifluoromethane Sulfonyl Ester ofTrimethylsilyloxy-PEG (Compound XI)

15.74 grams (10 mmol) of the crystalline 1-trimethylsilyloxy PEGobtained as described above (compound X) was dissolved in 40 mLanhydrous benzene and cooled in a bath of crushed ice. 1.53 mL (11 mmol)triethylamine and 1.85 mL (11 mmol) of trifluoromethanesulfonicanhydride obtained from Aldrich Chemical Co. were added and the mixturewas stirred overnight under an inert atmosphere until the reactionmixture changed to a brown color.

The solvent was then evaporated under reduced pressure and the residualsyrupy paste was diluted to 100.0 mL with methylene chloride. Due to thereactivity of trifluoromethane sulfonic esters, no further purificationof the trifluoromethane sulfonyl ester of 1-trimethylsilyloxy PEGcarried out.

C. Preparation of 1-Trimethylsilyloxy PEG 1500 PE Intermediate (CompoundXII)

10 mL of the methylene chloride stock solution of the trifluoromethanesulfonyl ester of 1-trimethylsilyloxy PEG (compound XI) was evaporatedto dryness under vacuum to obtain about 1.2 grams of residue(approximately 0.7 mmoles). To this residue, 3.72 mL of a chloroformsolution containing 372 mg (0.5 mmoles) egg PE was added. To theresulting solution, 139 microliters (1.0 mmole) of triethylamine wasadded and the solvent was evaporated under vacuum. To the residue wasadded 5 mL dry dimethyl formamide and 70 microliters (0.50 mmoles)triethylamine (VI). Air from the reaction vessel was displaced withnitrogen. The vessel was sealed and heated in a sand bath at 110° C. for22 hours. The solvent was evaporated under vacuum to obtain 1.58 gramsof brownish colored oil.

A 21×260 mm column filled with Kieselgel 60 silica gel, 70-230 mesh, wasprepared and wetted with a solvent composed of 40 volumes of butanone,25 volumes acetic acid and 5 volumes of water. The crude product wasdissolved in 3 mL of the same solvent and chromatographed using theabove-described solvent system. Sequential 30 mL portions of effluentwere each assayed by TLC.

The TLC analysis was carried out on silica gel coated glass plates usinga solvent combination of butanone/acetic acid/water; 40/25/5; v/v/v.Visualization was carried out using iodine vapor absorption. In thissolvent system, N-1-trimethylsilyloxy PEG-1500-PE appeared atR_(f)=0.78, Unreacted PE appeared at R_(f)=0.68.

The desired N-1-trimethylsilyloxy PEG 1500 PE was a chief constituent ofthe 170-300 mL portions of column effluent. When combined and evaporatedto dryness under vacuum, these portions afforded 111 mg of a pale yellowoil (1-trimethylsilyloxy-PEG-1500-PE intermediate).

D. Preparation of Polyethylene Glycol 1500: PE (Compound XII)

Once-chromatographed, the trimethylsilyloxy intermediate from Example 3Cabove was dissolved in 2 mL of tetrahydrofuran. To this, 6 mL aceticacid and 2 mL water was added. The resulting solution was allowed tostand for 3 days at 23° C. The solvent from the reaction mixture wasevaporated under vacuum and the resulting residue was dried to constantweight to obtain 75 mg of pale yellow wax. TLC on Si-C18 reversed-phaseplates eluted with a solvent mixture of 4:1 ethanol-water (v/v)indicated that some free PE and some polyglycol-like material formedduring the hydrolysis.

The residue was dissolved in 0.5 mL tetrahydrofuran and diluted with 3mL of a solution of 80:20 ethanol:water (v/v). The solution was appliedto the top of a 10 mm×250 mm chromatographic column packed withoctadecyl bonded phase silica gel and the crude product was eluted withan 80:20 ethanol:water (v/v) solvent system, collecting sequential 20 mLportions of effluent. The effluent was assayed by reversed phase TLC.Fractions containing product (Rf=0.08 to 0.15) were combined. Whenevaporated to dryness under vacuum these portions afforded 33 mg of acolorless wax (compound XII) corresponding to a yield of only 3%, basedon the starting phosphatidyl ethanolamine.

NMR analysis indicated that the product incorporated both PE residuesand PEG residues. The product was used to prepare PEG-PE liposomes.

E. Preparation of the Aldehyde of PEG-PE (Compound XIII)

The free hydroxyl group on PEG derivatized PE (compound XII) can beoxidized to the corresponding aldehyde in the following manner (Harris,J. M., J. Polym. Sci., Polym. Chem. Ed. 22:341-352 (1984)) prior toincorporation of the functionalized polymers into liposomes. About 2.7 gPEG1500-PE (1 mmole), prepared as in Example 3D, is added to 0.4 gacetic anhydride in 15 mL dimethylsulfoxide and the resulting mixture isstirred for 30 hours at room temperature. The reaction mixture is thenneutralized by addition of dilute sodium hydroxide and the solvent isevaporated under reduced pressure to yield a sticky residue.

The progress of the reaction may optionally be monitored by withdrawingaliquots of the reaction mixture, performing a mini work-up as describedbelow, and monitoring the appearance of an IR absorption correspondingto an aldehyde group.

The sticky residue is dissolved in 10 mL chloroform, washed with twosuccessive 10 mL portions of water, and the organic phase is dried overa drying agent such as anhydrous magnesium sulfate. Theproduct-containing chloroform phase is evaporated under vacuum to obtaina wax. The wax is then redissolved in 5 mL chloroform and purified bycolumn chromatography on silica gel using the following series ofsolvents: Volume % Volume % Methanol containing 2% Chloroform Conc.Ammonium Hydroxide/Methanol 100% 0% 95% 5% 90% 10% 85% 15% 80% 20% 70%30% 60% 40% 50% 50% 0% 100%

Typically, 50 mL fractions of column effluent are collected and analyzedby TLC on Si-C18 reversed-phase plates using a 4:1 ethanol:water (v/v)solvent system followed by I₂-vapor visualization.

Only those fractions containing an iodine-absorbing lipid with an Rfvalue of about 0.20 are combined and evaporated to dryness under vacuum,followed by drying under high vacuum to constant weight to yield 94 mgof a waxy crystalline solid product (compound XIII) with a molecularweight of 2226.

EXAMPLE 4 Synthesis of N-hydroxysuccinimide ester ofα-hydroxy-ω-(carboxymethylamino-carbonyl) PEG (Compound XXIV) andCoupling to DSPE

An α-hydroxy-ω-carboxy derivative of PEG (compound XIX) (2 g, ≈1 mmol)and N-hydroxysuccinimide (0.23 g, 2 mmol) were dissolved in methylenechloride-ethyl acetate (4 mL, 1:1). The resulting solution was cooled inan ice-water bath and treated with dicyclohexylcarbodiimide (DCC) (0.25g, 1.2 mmol) predissolved in ethyl acetate (1 mL). Within a few minutesthe solution became cloudy as dicyclohexylurea (DCU) appeared. After 2hours the reaction mixture was filtered to remove DCU and evaporated todryness. The functionalized polymer was crystallized from isopropanoland dried in vacuo over P₂O₅. Yield: 1.5 g (70%).

Titration of the product for active acyl content (Zaplipsky, S., et al.,POLYMERIC DRUGS (Dunn, R. L. and Ottenbrete, R. M., Eds.) AmericanChemical Society, pp. 91 (1991)) gave 4.8·10⁻⁵ mole/g (104% of thetheoretical value).

The N-hydroxysuccinimide ester of α-hydroxy-ω-carboxy-PEG (0.52 g, 0.2mmol) was added to a suspension of DSPE (0.14 g, 0.185 mmol) inchloroform (2 mL) followed by addition of triethylamine (0.1 mL, 0.86mmol). The mixture was heated in a water bath at 55° C. for 5 minutes,during which time the solution became clear. TLC(chloroform-methanol-water 90:18:2) on silica gel coated plates showedcomplete conversion of DSPE into a new product, which gave no color whentreated with ninhydrin. The solution was treated with an equivalentamount of acetic acid to neutralize the TEA and the neutralized solutionwas evaporated to dryness. The residue was dissolved in water andextensively dialyzed through a 300,000 MWCO cellulose acetate membraneat 4° C., filtered (pore size 0.2 μm) and lyophilized, yielding purecompound XXIV (360 mg, ≈70%).

This compound may then be further reacted with DSC to form aPEG-derivatized DSPE lipid containing an α-succinimidyl carbonate group.

EXAMPLE 5 Preparation of DSPE-PEG-Hydrazide (Compound XXXII)

A. Preparation of ω-Hydroxy Acid Derivative of PEG,α-(Hydroxyethyl)-ω-(carboxymethyl-aminocarbonyl)oxy-poly(oxyethylene)(Compounds XIX and XXIX)

Polyethylene glycol (Fluka, PEG-2000, 42 g, 42 mequiv OH) is dissolvedin toluene (200 mL), azeotropically dried (Zalipsky, S., et al., Int. J.Peptide Res. 30:740 (1987)) and treated with ethyl isocyanotoacetate(2.3 mL, 21 mmol) and triethylamine (1.5 mL, 10 mmol). The reactionmixture is stirred overnight at 25° C. and the solution is thenevaporated to dryness. The residue is dissolved in 0.2 M NaOH (100 mL)and any trace of toluene is removed by evaporation. The solution ismaintained at pH 12 with periodical dropwise addition of 4 M NaOH.

When the solution pH is stabilized at pH 12, the solution is acidifiedto pH 3.0 and the product is extracted with methylene chloride (100mL×2). TLC on silica gel (isopropyl alcohol/H₂O/conc. ammonia 10:2:1)gives a typical chromatogram of partially carboxylated PEG (Zalipsky, etal., 1990) consisting of unreacted PEG (R_(f)=0.67), monocarboxylatedderivative (R_(f)=0.55) and dicarboxylated derivative of the polymer(R_(f)=0.47). This solution is dried over anhydrous MgSO₄, filtered andevaporated to dryness. The PEG mixture is dissolved in water (50 mL).One-third of this solution (30 mL≈14 g of derivatized PEG) is loadedonto DEAE-Sephadex A-25 (115 mL of gel in borate form). After theunderivatized PEG is washed off the column with water (confirmed bynegative poly(methacrylic acid), PMA, test) (Zalipsky, et al., 1990), agradient of ammonium bicarbonate (2-20 mM at increments of 1-2 mM every200 mL) is applied, and 50 mL fractions are collected. Early elutingfractions, e.g., fractions 1-25, typically contain only PEG monoacid asdetermined by PMA and TLC analyses. These fractions are then pooled,concentrated to ≈70 mL, acidified to pH 2 and extracted with methylenechloride (50 mL×2). The CH₂Cl₂ solution is dried over anhydrous MgSO₄,concentrated and poured into cold stirring ether. The precipitatedproduct (compound XXIX) is dried in vacuo. Yield: 7 g. Titration ofcarboxyl groups gives 4.6·10⁻⁴ mequiv/g (97% of theoretical value).

B. Preparation of Compound XXX

Compound XXIX (5 g, 2.38 mmol) and tert-butyl carbazate (0.91 g, 6.9mmol) are dissolved in CH₂Cl₂-ethyl acetate (1:1, 7 mL). The solution iscooled on ice and treated with DCC (0.6 g, 2.9 mmol) predissolved in thesame solvent mixture. After 30 minutes the ice bath is removed and thereaction is allowed to warm to room temperature and stirred for anadditional 3 hours. The reaction mixture is filtered to removedicyclohexylurea and the resulting filtrate is evaporated to produce acrude residue. The residue is recovered and purified by twoprecipitations from ethyl acetate-ether (1:1) and dried in vacuo overP₂O₅. Yield: 5.2 g, 98%. TLC of the product reveals one spot(R_(f)=0.68) with an R_(f) value different from that of the startingmaterial (R_(f)=0.55). H-NMR (CDCl₃): δ 1.46 (s, t-Bu, 9H); 3.64 (s,PEG, 178H) 3.93 (br. d, J=4.5, CH₂ of Gly, 2H); 4.24 (t, CH ₂—OCO-Gly,2H) ppm. ¹³C-NMR (CDCl₃): δ 28.1 (t-Bu); 43.4 (CH₂ of Gly); 61.6(CH₂OH); 64.3 (CH₂OCONH); 69.3 (CH₂CH₂OCONH); 70.5 (PEG); 72.4(CH₂CH₂OH); 81.0 (CMe₃); 155.1 (C═O of Boc); 156.4 (C═O of Gly urethane;168.7 (C═O of Gly hydrazide) ppm.

C. Preparation of Compound XXXI

The ω-hydroxy Boc-hydrazide derivative of PEG (compound XXX, 5 g, 2.26mmol) is dissolved in pyridine (1.1 mL), CH₂Cl₂ (5 mL) and CH₃CN (2 mL)and treated with disuccinimidyl carbonate, DSC (1.4 g, 5.5 mmol). Thereaction mixture is stirred at 25° C. overnight. The mixture is thenfiltered to remove solids and slowly added to cold ethyl ether (100 mL).The precipitated product is dissolved in warm ethyl acetate (45 mL),chilled and mixed with equal volume of ethyl ether. The precipitate iscollected by filtration and dried in vacuo over P₂O₅. Yield of compoundXXXI: 4.8 g, 90%.

Succinimidyl carbonate group content 4.15·10⁻⁴ mequiv/g (98% oftheoretical value) is determined by titration (Zalipsky, et al., 1991).H-NMR (CDCl₃): δ 1.46 (s, t-Bu, 9H); 2.83 (s, succinimide); 3.64 (s,PEG, 178H); 3.79 (t, CH ₂CH₂OCO₂-Su); 3.93 (br. d, J=4.5, CH₂ of Gly,2H); 4.24 (t, CH ₂—OCO-Gly, 2H); 4.46 (t, CH ₂OCO₂-Su) ppm.

D. Preparation of Compound XXXII

To prepare the DSPE-PEG-hydrazide, a slight excess of succinimidylcarbonate Boc-protected PEG-glycine hydrazide (compound XXXI) is reactedwith DSPE suspended in chloroform in the presence of triethylamine. Thelipid derivative is quickly (5-10 minutes) solubilized during progressof the reaction. Excess heterobifunctional PEG is removed by dialysisusing a 300,000 MWCO cellulose ester dialysis membrane from Spectrum.The recovered lipid conjugate is subjected to conventionalBoc-deprotection conditions (4M HCl in dioxane for 30 minutes) and thenfurther purified by recrystallization. H-NMR (CDCl₃): δ 0.88 (t, CH₃,6H); 1.59 (t, CH ₂CH₂CO, 4H); 2.84 (t, CH₂CO, 4H); 3.64 (s, PEG, 180H);4.0 (t); 4.2 (m, CH₂OCO—NH₂); 4.4-4.3 (two doublets); 5.2 (g, CH ofglyceride).

EXAMPLE 6 Preparation of Liposomes with Covalently Bound β-Galactosidase

The maleimide of the DSPE carbamide of polyoxyethylene bis(amine)(3500-DSPE) was prepared as in Example 1. β-Galactosidase was purchasedfrom Pierce (Rockford, Ill.). Enzyme assays with o-nitrophenyl galactosewere performed essentially by monitoring the development of the coloredproduct with an extinction coefficient of 4467 at 413 nanometers in 0.1N sodium hydroxide. The assay mixture consisted of 86 mM sodiumphosphate pH 7.3, 1 mM magnesium chloride, 50 mM beta-mercaptoethanoland 2.3 mM o-nitrophenyl galactose and product formation was monitoredfor 10 to 15 minutes in the linear range of the assay.

Liposomes (MLV's) were prepared according to standard methods with oneof the compositions indicated in Table 4. The liposomes were sized byextrusion through a polycarbonate membrane to 200 nm. TABLE 4“Phenotype” Mol % PEG- PEG- DSPE Crosslinker αT Ch Pc Crosslinker DSPEPG − − 1 33 61 − − 5 + − 1 33 61 − 5 − − + 1 33 56 5 − 5 + + 1 33 56 5 5−where α-T=α-tocopherol (antioxidant), Ch=cholesterol, PC=partiallyhydrogenated egg PC (IV 40), crosslinker=the maleimide derivative ofPEG-3500-DSPE, and PG=egg phosphatidyl glycerol. In addition, allliposome preparations were “spiked” with a ³H-DPPC tracer. The totallipid concentration in each preparation, after hydration in PBS (50 mMsodium phosphate pH 7.2, 50 mM sodium chloride, was 2 mM.

Crosslinking reactions were performed by adding enzyme solution to theliposomes (final protein concentration=0.5 mg/mL) and incubating thesuspension overnight at ambient temperature with gentle shaking.Unreacted crosslinker was then quenched with 10 mM 2-mercaptoethanol(2-ME) for 30-60 minutes at 37° C. Liposomes were separated fromunconjugated protein by flotation through a metrizamide gradient: thesample was brought to 30% (w/v) metrizamide and transferred to an SW60Titube, 20% metrizamide was layered above, then PBS was added on top toprovide an aqueous interface. Gradients were centrifuged at 45,000 rpmfor 60 minutes at 4° C., then each liposomal band, easily visible at thePBS interface, was collected and transferred to dialysis tubing.Dialysis proceeded overnight at 4° C. against two changes of PBS.Removal of the metrizamide was necessary because it inhibitsβ-galactosidase activity significantly even at 1% (w/v) concentration.

EXAMPLE 7 Liposome Blood Lifetime Measurements of HydrazideEnd-functionalized PEG Liposomes

A. Preparation of Hydrazide End-functionalized Liposomes

Hydrazide PEG-DSPE composed of PEG, end-functionalized with a hydrazidegroup, and distearyl-PE was prepared as described. The hydrazidePEG-DSPE lipid was combined with partially hydrogenated egg PC (PHEPC)and cholesterol in a lipid:lipid:lipid mole ratio of about 0.15:1.85:1and the lipid mixture was hydrated. Generally, lipid hydration occurredin the presence of desferal mesylate, followed by sizing to 0.1 micron,and removal of non-entrapped desferal by gel filtration with subsequentloading of Ga-oxide into the liposomes. The unencapsulated Ga wasremoved during passage through a Sephadex G-50 gel exclusion column.Both compositions contained 10 micromoles/mL in 0.15 M NaCl, 5 mMdesferal.

A second lipid mixture was prepared in a similar manner but with HSPC(hydrogenated serum phosphatidylcholine) instead of PHEPC.

B. Measuring Blood Circulation Time and Tissue Levels

In vivo studies of liposomes were performed in laboratory rats weighing200-300 g each. These studies involved tail vein injection of liposomesamples at about 10-20 micromolar phospholipid/kg body weight. Bloodsamples were obtained by retroobital bleeding at defined times. Theanimals were sacrificed after 24 hours and tissues removed for labelquantitation. The weight and percent of the injected dose in each tissuewas determined. The studies were carried out using ⁶⁷Ga-desferal loadedliposomes and radioactivity was measured using a gamma counter. Thepercent of the injected dose remaining in the blood at several timepoints up to 24 hours, and in selected tissues at 24 hours wasdetermined as follows.

1. Plasma Kinetics of Hydrazide-PEG Liposomes.

The above-described liposome composition (0.4 mL) was intravenouslyadministered and at times 0, 0.25, 1, 3, or 5 and 24 hours afterinjection, blood samples were removed and assayed for the amount ofGa-desferal present in the blood, expressed as a percentage of theamount measured immediately after injection.

Hydrazide-PEG liposomes have a blood halflife of about 15 hours, andnearly 30% of the injected material was determined to be present in theblood after 24 hours.

2. 24 Hour Tissue Levels

Studies to determine the distribution of gallium-labelled liposomes inselected tissues 24 hours after intravenous injection were performed.The liposome composition (0.4 mL) was intravenously administered asdescribed in 7B above. The percent dose remaining in tissues 24 hoursafter intravenous administration is shown in Table 3.

While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications and changes may be made without departing from theinvention.

1. A liposome composition, comprising liposomes, each having an outerlayer of a hydrophilic, and an effector molecule attached to the distalends of said chains, said effector molecule having binding affinity to acell receptor, wherein said liposome-bound effector molecule binds tothe cell receptor and sterically hinders the cell receptor.
 2. Thecomposition of claim 1 wherein the effector molecule is selected fromthe group consisting of F_(ab) antibody fragments, cytokines, cellulargrowth factors, peptide hormones, monosaccharides, polysaccharides, IL-1inhibitors, ELAM-1 binding inhibitors, and limulusantilipopolysaccharide factor (LALF).
 3. The composition of claim 2wherein the polysaccharide is sialyl Lewis^(x).
 4. The composition ofclaim 2 wherein the cytokine is selected from the group consisting ofinterferons, interleukins, TNF, transforming growth factor β,lymphotoxin, GM-CSF, and G-CSF.
 5. The composition of claim 4 whereinthe interferon is selected from the group consisting of IFN-alpha,IFN-beta, and IFN-gamma.
 6. The composition of claim 4 wherein theinterleukin is selected from the group consisting of IL-1α, IL-1β, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, and IL-8.
 7. A liposome composition foruse in treating a condition mediated by binding of one binding member toa second binding member, comprising liposomes, each having an outerlayer of a hydrophilic polymer, an effector molecule attached to thedistal ends of said chains, said effector molecule having bindingaffinity to a cell receptor, wherein said liposome-bound effectormolecule binds to the cell receptor and sterically hinders the cellreceptor.
 8. The composition of claim 7 wherein the effector molecule isselected from the group consisting of F_(ab) antibody fragments,cytokines, cellular growth factors, peptide hormones, monosaccharides,polysaccharides, IL-1 inhibitors, ELAM-1 binding inhibitors, and limulusantilipopolysaccharide factor (LALF).
 9. The composition of claim 8wherein the polysaccharide is sialyl Lewis^(x).
 10. The composition ofclaim 8 wherein the cytokine is selected from the group consisting ofinterferons, interleukins, TNF, transforming growth factor β,lymphotoxin, GM-CSF, and G-CSF.
 11. The composition of claim 10 whereinthe interferon is selected from the group consisting of IFN-alpha,IFN-beta, and IFN-gamma.
 12. The composition of claim 10 wherein theinterleukin is selected from the group consisting of IL-1α, IL-1β, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, and IL-8.